1 - Informatics

1.Protein expression and purification
ORF cloning Recombinational strategies for rapid and
precise cloning of amplified DNA
A) Gap repaired mediated recombination in yeast
B) ligation independent cloning
C) Recombination by the gateway cloning system
It can take one month today to clone all the ORF of a
2.Affinity tags: Tag affect expression, yield and purity when
recombinant proteins are expressed in E. Coli.
GST: Gluthatione S transferase is fused to the protein. Bind
to gluthathione agarose columns and eluted with guthathione
Polyhistidine: Tag made of 6 polyhistidines at the N ter of
Cter of the protein retained on nickel agarose column elution
with imidazole.
Maltose binding protein: Allows better solubilization of the
Cellulose binding protein
Calmodulin-binding peptides: bind to calmodulin agarose
column and are eluted with the chelator EGTA.
Myc and flag tag are used for immunoprecipitation and
detection by western blot.
Green fluorescent protein are used to detect the localization
of the protein in living cells and for the detection of protein
interaction through fluorescence energy transfer.
3.Protein complex
Two proteins become in contact
The recognition area define the interface
Protein complexes are formed by shape and charge
complementarity with or whitout conformational change.
As rigid bodies (non conformational change) they generally
have a standard interface
less than 2000A2
With conformational changes they generally form larger
More than 2000 A2
Large interface involve more than one patch of recognition.
involved in protein-protein interface can be hydrophobic,
and/or electrostatics
Protein complexes can be transient of stable.
4.Yeast 2-hybrid system
The yeast two-hybrid system. DNA-binding and activation domains (circles)
are fused to proteins X and Y; the interaction of X and Y leads to reporter gene
expression (arrow).
b) A standard two-hybrid search. Protein X, present as a DNA-binding domain
hybrid, is screened against a complex library of random inserts in the activationdomain vector (square brackets).
c) A two-hybrid array approach. Protein X is screened against a complete set of
full-length open reading frames (ORFs) present as activation-domain hybrids
(shown as yeast transformants spotted onto microtitre plates).
d) A two-hybrid search using a library of full-length ORFs. The set of ORFs as
activation-domain hybrids (microtitre plates in square brackets) is combined to
form a low-complexity library.
e) A two-hybrid pooling strategy. Pools of ORFs as both DNA-binding domain
and activation-domain hybrids (square brackets) are screened against each other.
5.Mass spectrumetry
Tandem Affinity Purification
high-throughput mass-spectrometric protein complex identification
Bait proteins are tagged with a Flag epitope
Excision and digestion of the bands with trypsin
MS/MS Protein identification
Protein identification via MS/MS spectrometry